NIGMS-Supported Basic Research on Skin Replacement Following Burn or Trauma Injury

by Alison Davis
June 18, 1999

Our protective armor, skin, is the largest organ in the body. But armor only, skin is not. This highly dynamic network of cells, nerves, and blood vessels serves the body in diverse ways.

Clearly, skin's protective function is paramount, providing internal organs and tissues with a physical barrier from the environment and the dangers therein: toxins, heat and cold, and disease-carrying microbes. But skin also plays an important role in preserving fluid balance and in regulating body temperature and sensation. Nerves buried deep within skin allow us to sense the presence of potentially harmful invaders, such as bees. Immune cells resident in skin help the body prevent and fight disease.

For these reasons, the loss of skin due to burns or trauma can deal the body a severe blow, impairing or even eliminating the many vital functions this organ performs.

Burns

Each year in the United States, 1.1 million burn injuries demand medical attention. Ten thousand people die every year of burn-related infections. Tragically, many burn victims are children. The good news is that, in recent years, survival statistics for serious burns have improved dramatically. Twenty years ago, for instance, burns covering half the body were routinely fatal. Today, patients with burns encompassing 90 percent of their body surface can survive, albeit sometimes with permanent impairments.

By funding basic research aimed at understanding how the body, especially skin, responds to burn- and trauma-related injury, the National Institute of General Medical Sciences (NIGMS) has played an important role in driving burn injury survival statistics upward. Among the advances that have contributed directly to this public health benefit are discoveries of the importance of proper wound care, adequate nutrition, and infection control. NIGMS-funded research has also led to the development of widely used commercially available skin-replacement products for the treatment of injury caused by severe burns.

Burn-induced skin loss affords bacteria and other microorganisms easy access to the warm, moist, nutrient-rich fluids that course through the body, while at the same time it provides a conduit for the rapid and dangerous loss of these fluids. Extensive blood loss can thrust a burn or trauma victim into shock, a life-threatening condition in which blood pressure plunges so low that vital organs--such as the brain, heart, and kidneys--simply cannot get enough blood (and thereby oxygen) to function. Hence, replenishing skin lost to severe burns is an urgent matter in the care of a burn patient. When a patient has lost 80 or 90 percent of the skin as a result of direct contact with scalding hot liquids, flames, harsh chemicals, electrical current, or nuclear radiation, two immediate tasks come to the fore.

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First, a burn surgeon must surgically remove the burned skin, then the unprotected underlying tissue must be quickly covered. Two classes of biomaterials useful in covering the wound are laboratory-grown skin cells and artificial skin; the two are sometimes used in combination.

Laboratory-Grown Skin Cells

In the mid-1980s, with a grant from NIGMS, Dr. Howard Green of Harvard Medical School conceived a method for growing a type of human skin cells called keratinocytes (which populate skin's upper, or epidermal, layer) outside of the body. Dr. Green's keratinocyte culture research paved the way for the method to proceed to commercialization.

Growing cells in a laboratory, a technique called "culturing," can be a tricky business. No general recipe exists: Every different cell type in the body requires a unique set of conditions, and some simply will not grow this way at all. The secret to Dr. Green's technique, in which he "seeded" human keratinocytes onto a layer of mouse-derived fibroblast (connective tissue) cells in a plastic culture dish, is derived from Mother Nature herself. Presumably, the technique works because it mimics what happens in actual skin, whose lower layer, called the dermis, is composed predominantly of fibroblasts. The principle function of fibroblasts is to produce proteins called collagen and elastin that provide structure to skin. To ensure that the keratinocytes, not the fibroblast "feeder" cells, would multiply in his culture flask, Dr. Green first irradiated the fibroblasts so they would not continue to divide but would still pump out nutrients into the culture broth. After several days in such an environment, the few starting keratinocytes grew into a sheet of epidermal-like tissue.

The product that eventually resulted from Dr. Green's work, called Epicel™, is currently licensed by a company called Genzyme Tissue Repair (Cambridge, Massachusetts). Epicel™ is used to treat deep wounds that require grafting (skin replacement), such as occurs with severe burns. However, since Epicel™ replaces the lost epidermal layer only, it works best in combination with something that restores the dermal layer of skin. Epicel™ is not an artificial skin, but rather a method in which new epidermis is "grown to order" in a laboratory from surgically harvested skin cells taken from an unburned area of the patient. Products like Epicel™ are termed "autologous" grafts, meaning that the source of the epidermal graft material is taken from skin of the same patient who receives it. (In contrast, the source of skin for an "allograft" is skin from another person, sometimes even a cadaver. Allografts offer only temporary cover, as they are quickly rejected by the patient's immune system.)

Artificial Skin

In very severely burned patients who have little or no remaining intact skin, artificial skin is an extremely useful material not only to cover and thereby protect the wounded area, but to promote re-growth of a natural skin instead of scar tissue. NIGMS-funded studies begun in the early 1970s and continuing into the 1980s led to clinical testing and commercial production of an artificial skin system called Integra® Dermal Regeneration Template™. Every similar artificial skin product that has since been researched and developed hinges upon the conceptual framework that eventually yielded this artificial skin system.

The brainchild of a trauma surgeon and a mechanical engineer, Integra® is a prime example of NIGMS' investment in collaborative research. In the early 1970s, the surgeon, Dr. John F. Burke, then director of the Burn Center at Massachusetts General Hospital and Shriners Burns Institute, came up with the idea that completely removing badly burned skin (as opposed to letting it slough off over time) might offer greater protection against wound infection and improve the very poor prognosis that severely burned patients faced. Dr. Burke recognized that a necessary follow-up to the removal of burned skin would be immediate and permanent skin replacement. Once developed, his idea ultimately became standard practice for treating major burn injuries.

At first, Dr. Burke pioneered the use of skin from related donors (such as family members with similar genetic markers). But doing so required that the burn patient be given powerful immunosuppressant drugs, to dampen the patient's immune system so that the graft would not be rejected. Unfortunately, crippling the immune system in this way posed many serious problems for the patient. Instead, Dr. Burke began using the patient's own unburned skin (often from the scalp, which is rarely burned) as a source of graft material.

However, since using these sorts of grafts (or even skin from cadavers) did not permanently solve the problem, Dr. Burke saw the need for some type of artificial means to recover skin. Using a synthetic product would also offer an advantage in that such a material is free of viruses and bacteria, which can transmit disease. Dr. Burke, who had a penchant for engineering, recruited a clever mechanical engineer at neighboring MIT, Dr. Ioannas Yannas, to cooperate in this effort. The collaboration, marrying biomedical engineering with clinical medicine, proved fruitful. After initial testing in animals, the artificial skin that Drs. Burke and Yannas developed proceeded to rigorous scientific testing in humans, in a multi-center clinical trial.

Integra® contains no living components, and it is not itself actually designed to be a replacement skin. Rather, it supplies a protective covering and a pliable scaffold onto which the patient's own skin cells can "regenerate" the lower, dermal layer of skin that was destroyed by the burn. Actually, unlike analagous skin cells of certain amphibians and other types of animals, human dermis cannot regenerate in the true sense. In addition to fibroblasts and other cells, human dermis also contains hair follicles, sweat glands, and networks of blood vessels. As it contains so many diverse components arranged just so, the dermal layer is nearly impossible to replicate. What Drs. Burke and Yannas discovered, however, is that remaining fibroblasts deep within a badly burned skin wound can be "instructed" on how to arrange themselves into something resembling a real dermis. Basically, the trick is to coax those existing fibroblasts and other supporting cells into a pattern that resembles normal, healthy skin and not a scar.

Proper patterning of the many components of skin is a vital feature permitting this tissue to carry out its multiple tasks for the body.

Integra® consists of two layers, just as living skin is structured. The bottom layer, designed to "regenerate" the lower, dermal layer of real skin, is composed of a matrix of interwoven bovine collagen (a fibrous cow protein) and a sticky carbohydrate (sugar) molecule called glycosaminoglycan that mimics the fibrous pattern of dermis. This matrix is then affixed to a temporary upper layer: a medical-grade, flexible silicon sheet that mimics the epidermal, or surface, layer of skin. The product looks somewhat like translucent plastic wrap. After first removing tissue destroyed by the burn, a burn surgeon drapes Integra® over the wounded area of the patient and leaves it there for 2 to 4 weeks, during which time the patient's own cells make their way into the matrix and create a new dermis. The top layer of Integra® is then removed, and a very thin sheet of the patient's own epithelial cells is applied. Over time, a normal epidermis (except for the absence of hair follicles) is reconstructed from these cells. Key features of this material's design are the number and size of the holes in the collagen/glycosaminoglycan matrix as well as the rate at which the matrix disintegrates. The precise balance of these two components allows real skin to take hold.

Integra® was originally licensed, tested, and produced by Marion Laboratories of Kansas City, Missouri and is now being manufactured and sold by Integra LifeSciences Corporation of Plainsboro, New Jersey. NIGMS currently funds this company, through a Federal Small Business Innovation Research grant, to investigate what potential benefits adding a molecule that coaxes new growth of blood vessels within the original matrix might add to the quality and/or speed of skin regeneration of a burn-wounded area.

Another product resulting from NIGMS-sponsored research that is similar to Integra® is called AlloDerm™. This product, which is sold and manufactured by LifeCell Corporation of The Woodlands, Texas, is produced by removing from cadaver skin all cell components that cause a burn patient's immune system to reject a graft from any other person. The principle behind the product AlloDerm™ got its start in the mid-1980s in the laboratory of Dr. Charles Baxter of the University of Texas Southwestern Medical Center at Dallas, as well as in other laboratories working in this area of research. A key feature of the process is preserving to the greatest extent possible the "natural," three-dimensional structure of the dermis. Properly approximating this scaffold, whether from real dermis (as in AlloDerm™) or artificial dermis (as in Integra®), is crucial to the ability of the patient's remaining cells to regenerate themselves into a new, functioning skin.

On the Horizon

Since burn and trauma victims endure injuries that affect many body systems besides skin, NIGMS sponsors research aimed at understanding the complex, multi-organ response to injury caused by trauma or burns. For example, research on smoke inhalation injury, which causes the majority of burn patient deaths, has led to changes in treatment practices in burn units. NIGMS-funded work by Dr. Daniel Traber of the University of Texas Medical Branch at Galveston and Dr. Robert Demling of the Burn Unit at Brigham and Women's Hospital in Boston has shown that the right combination of aerosolized medications and hormones, delivered straight into the lungs, can significantly decrease airway damage due to smoke inhalation.

Proper nutrition may seem far from the minds of a critically injured burn patient or his or her doctor. But delivering the wrong mix of nutrients and minerals into the bloodstream can do more harm than good. NIGMS-funded research by Dr. David Herndon of the University of Texas Medical Branch at Galveston contributed to this conclusion by showing that making the intestinal tract "work" (by feeding the patient by mouth, instead of intravenously) keeps bacteria that normally live in the stomach from seeping into the bloodstream and causing body-wide infections that often lead to deadly septic shock.

Finally, NIGMS is funding other research on skin replacement, including a strategy to remove keratinocytes from non-burned epidermal skin, grow the keratinocytes into large sheets of cells in a laboratory, then place the sheets of cells on top of a collagen-based matrix that has been bathed in a nutritious mix of growth factors. When the material is grafted onto a patient, these factors prod the growth of new blood vessels. Dr. Steven Boyce of the University of Cincinnati and the Cincinnati Shriner's Burns Hospital has succeeded in expanding a small number of skin cells into a transplantable sheet that can be layered on top of Integra®. Dr. Boyce has assessed this method in a small number of patients, and he has obtained promising preliminary results that the method offers a significant advantage over other currently available technologies.

References

Burke JF, Bondoc CC, Quinby WC. Primary Burn Excision and Immediate Grafting: A Method of Shortening Illness. J Trauma 1974;14:389-95.

Burke JF, Quinby WC, Bondoc CC. Primary Excision and Prompt Grafting as Routine Therapy for the Treatment of Thermal Burns in Children. Surgery Clinics of North America 1976;56:477-94.

Green H, Kehinde O, Thomas J. Growth of Cultured Human Epidermal Cells into Multiple Epithelia Suitable for Grafting. Proc Natl Acad Sci USA 1979;76:5665-8.

Burke JF, Yannas IV, Quinby WC Jr, Bondoc CC, Jung WK. Successful Use of a Physiologically Acceptable Artificial Skin in the Treatment of Extensive Burn Injury. Ann Surg 1981;194:413-28.

Yannas IV, Burke JF, Orgill DP, Skrabut EM. Wound Tissue Can Utilize a Polymeric Template to Synthesize a Functional Extension of Skin. Science 1982;215:174-6.

Gallico GG III, O'Connor NE, Compton CC, Kehinde O, Green H. Permanent Coverage of Large Burn Wounds with Autologous Cultured Human Epithelium. N Engl J Med 1984;311:448-51.

Heck EL, Bergstresser PR, Baxter CR. Composite Skin Graft: Frozen Dermal Allografts Support the Engraftment and Expansion of Autologous Epidermis. J Trauma 1985;25:106-12.

Thompson PB, Herndon DN, Traber DL, Abston S. Effect on Mortality of Inhalation Injury. J Trauma 1986;26:163-5.

Tompkins RG, Burke JF, Schoenfeld DA, Bondoc CC, Quinby WC Jr, Behringer GC, Ackroyd FW. Prompt Eschar Excision: A Treatment System Contributing to Reduced Burn Mortality. A Statistical Evaluation of Burn Care at the Massachusetts General Hospital (1974-1984). Ann Surg 1986;204:272-81.

Heimbach D, Luterman A, Burke J, Cram A, Herndon D, Hunt J, Jordan M, McManus W, Solem L, Warden G, Zawacki B. Artificial Dermis for Major Burns: A Multi-Center Randomized Clinical Trial. Ann Surg 1988;208:313-20.

Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ. Burn Wound Closure with Cultured Autologous Keratinocytes and Fibroblasts Attached to a Collagen-Glycosaminoglycan Substrate. JAMA 1989;262:2125-30.

Herndon DN, Barrow RE, Stein M, Linares H, Rutan TC, Rutan R, Abston S. Increased Mortality with Intravenous Supplemental Feeding in Severely Burned Patients. J Burn Care Rehab 1989;10:309-13.

Kramer GC, Herndon DN, Linares HA, Traber DL. Effects of Inhalation Injury on Airway Blood Flow and Edema Formation. J Burn Care Rehab 1989;10:45-51.

Tompkins RG, Hilton JF, Burke JF, Schoenfeld DA, Hegarty MT, Bondoc CC, Quinby WC Jr, Behringer GE, Ackroyd FW. Increased Survival After Massive Thermal Injuries in Adults: Preliminary Report Using Artificial Skin. Crit Care Med 1989;17:734-40.

Stern R, McPherson M, Longaker MT. Histologic Study of Artificial Skin Used in the Treatment of Full-Thickness Thermal Injury. J Burn Care Rehab 1990;11:7-13.

Boyce ST, Goretsky MJ, Greenhalgh DG, Kagan RJ, Rieman MT, Warden GT. Comparative Assessment of Cultured Skin Substitutes and Native Skin Autograft for Treatment of Full-Thickness Burns. Ann Surg 1995;222:743-52.

Boyce ST, Kagan RJ, Meyer NA, Yakuboff KP, Warden GD. Cultured Skin Substitutes Combined with Integra® to Replace Native Skin Autograft and Allograft for Closure of Full-Thickness Burns. J Burn Care Rehab 1999;in press.

Relevant Grants and Funding Periods

GRANT	FUNDING PERIOD	PRINCIPAL INVESTIGATOR	INSTITUTION
Burn Trauma Center P50 GM21700	1974-present	John Burke, then Ronald Tompkins	Massachusetts General Hospital
Research in Burns and Trauma T32 GM07035	1975-present	John Burke, then Ronald Tompkins	Massachusetts General Hospital
Design and Evaluation of an Artificial Skin R01 GM23946	1977-1989	Ioannis Yannas	Massachusetts Institute of Technology
Pathophysiologic, Biochemical Changes of Thermal Injury P50 GM21681	1978-present	Charles Baxter	University of Texas Southwestern Medical Center at Dallas
Pathophysiology of Lungs After Thermal Injury R01 GM31662	1982-present	Robert Demling	Brigham and Women's Hospital/Beth Israel-Deaconess Medical Center
Living Prosthetic Skin R01 GM31465	1983-1985	John Hansbrough	University of Colorado Health Sciences Center
Regeneration of Epidermis by Grafting Cultured Cells R01 GM33158	1984-1989	Howard Green	Harvard University
Living Prosthetic Skin R01 GM35068	1985-1988	John Hansbrough	University of California, San Diego
Smoke Inhalation Injury R01 GM33324	1985-1996	Daniel Traber	University of Texas Medical Branch at Galveston
Burn/Trauma Research Training Grant T32 GM08247	1988-1993	John Hansbrough	University of California, San Diego
Postdoctoral Training in Trauma and Burns T32 GM08256	1990-present	David Herndon	University of Texas Medical Branch at Galveston
Mechanisms of Wound Healing with Cultured Skin R01 GM50509	1994-present	Steven Boyce	University of Cincinnati
Modulation of the Postburn Hypermetabolic Response R01 GM56687	1998-present	David Herndon	University of Texas Medical Branch at Galveston
Peptide Enhanced Artificial Skin R44 GM59531	Awarded in 1999	Frederick Cahn	Integra LifeSciences Corporation